In recent years, there has been an explosion of information on the new possibility of inducing immunity using nucleic acids, called DNA vaccination. Previous studies have shown that plasmid DNA introduced into an adult immunocompetent host could induce an antibody response (Tang et al., Nature 356:152-154 (1992)), it was soon demonstrated using the influenza virus that both humoral and cell-mediated could be induced, and these were sufficient for protection in vivo (Ulmer et al., Science 259:1745-1749 (1993); Fynan et al., Proc. Natl. Acad. Sci. USA 90:11478-11482 (1993)). It soon became evident that, DNA vaccines, also called genetic vaccines, have gone through a phase of exponential growth and found an application against a large variety of antigens. They have been applied to immunize against cancer (Conry et al., Cancer Res. 54:1164-1168 (1994); bacteria (Tascon et al., Nat. Med. 2:888-892 (1996); Huygen et al., Nat. Med. 2:893-898 (1996)); virus (Ulmer et al., supra, 1993; Fynan et al., supra, 1993; Raz et al., Proc. Natl. Acad. Sci. USA 91:9519-9523 (1994); Davis et al., Vaccine 12:1503-1509 (1994); Wang et al., Proc. Natl. Acad. Sci. USA 90:4156-4160 (1993); and parasites (Sedegah et al., Proc. Natl. Acad. Sci. USA 91:9866-9870(1994)).
This basis of DNA vaccination is the introduction into a host of the “blue-print” for vaccine molecules in a way that mimics viral infections without the infectious threat. The use of DNA vaccines can be of particular value in those instances in which vaccines are either ineffective or not yet available.
Genetic vaccines, while conceptually simple in their making, for example, a bacterial plasmid incorporating the coding region of interest, they are still fraught with a number of unresolved issues pertaining mainly to their biology at the site of injection and systemically. Genetic vaccines work along the same lines of recognition of antigen (B cells) and antigen processing and presentation (T cells) known and established for protein antigens or infectious pathogens.
It has been known for many years that eukaryotic DNA is per se scarcely immunogenic whereas prokaryotic (bacterial) DNA possesses properties relevant to immunogenicity. It has been known for some time that bacterial DNA could serve as adjuvant in immunization (Braun et al., Proc. Soc. Exp. Biol. Med. 119:701 (1965)). It was subsequently reported that mice immunized with Escherichia coli DNA complexed with methylated BSA in adjuvant produce significantly greater amounts of antibodies than mice immunized with calf thymus DNA (Gilkeson et al., Clin. Immunol. Immunopathol. 51:362-371 (1989); Gilkeson et al., J. Immunol. 142:1482-1486 (1989)). Earlier observations had already shown that bacterial DNA possesses immunostimulatory properties (Tokunaga et al., J. Natl. Cancer Inst. 72:955-962 (1984), a fact undisputed today (Messina et al., Cell Immunol. 147:148-157 (1993)). These stimulatory properties are apparently linked to a six-base nucleotide motif consisting of an unmethylated CpG dinucleotide (Krieg et al., Nature 374:546-549 (1995) expressed nearly twenty times more frequently in bacterial than in vertebrate DNA (Cardon et al., Proc. Natl. Acad. Sci. USA 91:3799-803 (1994)). Noncoding, immunostimulatory sequences (ISS)-enriched plasmid DNA or ISS oligonucleotides themselves stimulate immune responses to co-administered antigens (Roman et al., Nat. Med. 3:849-854 (1997)) by activating IFN-g, IL-12 and IL-18, all of which promote a Th1 response (Carson and Raz, J. Exp. Med. 186:1621-1622 (1997)).
In 1992, plasmid DNA was shown to immunize against the very antigen it codes for (Tang et al., Nature 356:152-154 (1992)). Therefore, it was possible to immunize using foreign DNA controlled by a potent promoter for tissue expression. Inoculation of functional genes into somatic cells of adult immunocompetent animals is a simple way to mimic natural infection and initiate adaptive immunity (Ulmer et al., Curr. Opin. Immunol. 8:531-536 (1996)). Plasmid DNA containing antigen-coding sequences and regulatory elements for their expression can be introduced in tissues by parenteral injection (Wang et al., supra, 1993) or by particle bombardment (Tang et al., supra, 1993). Antibody production (B-cell immunity), and cell-mediated immunity of the helper or cytotoxic T cell-type, have been induced against viruses (Ulmer et al., supra, 1993), bacteria (Huygen et al., supra, 1996; Tascon et al., supra, 1996), parasites (Sedegah et al., supra, 1994), tumor antigens (Conry et al., supra, 1994), self antigens (Gilkeson et al., J. Exp. Med. 183:1389-1397 (1996); Waisman et al., Nat. Med. 2:899-905 (1996)) and allergens (Raz et al., Proc. Natl. Acad. Sci. USA 93:5141-5145 (1996)).
Typically, injections of plasmid DNA via the intramuscular or intradermal route yields both antibody and cellular responses with long-lasting immunity preferably induced by multiple DNA inoculations (Sedegah et al., supra, 1994; Xiang et al., supra, 1994). The transgene product is, however, rarely found in the circulation (Davis et al., supra, 1993), and little is known about where and how antigen presentation occurs.
From a practical stand-point, immunization via DNA inoculation relies on in vivo transfection, production and possibly secretion of the transgene product, and antigen presentation by specialized cells. However, in most studies, neither the in vivo transfected cells nor the antigen presenting cells involved in this process have been identified. Expression of foreign DNA under the control of viral promoters (Tang et al., supra, 1992; Ulmer et al., supra, 1993; Davis et al., supra, 1993; Raz et al., supra, 1994; Wang et al., supra, 1993; Huygen et al., supra, 1996; Tascon et al., supra, 1996; Sedegah et al., supra, 1994; Dollan et al., supra, 1996) limit tissue specificity. Therefore, no control of expression is possible other than the site of DNA inoculation.
Although genetic vaccines have been used successfully, there remains a need to develop more effective methods to exploit the immunogenic potential of genetic vaccines. The present invention satisfies this need and provides related advantages as well.